In Vivo MR Tracking Of Transplanted Spio-labeled Bone Marrow Stem Cells In Vitro And Stransplantation For Treating Traumaic Brain Injury In Rat

ObjectiveIn this study, bone marrow stem cells (BMSCs) were isolated, cultured and labled with different concentration of superparamagnetic iron oxid (SPIO) in vitro, Aimed at clarifying the activity of the labled cells with different concentration of SPIO; To observe the characteristics of the labled cells in different MR sequences in vivo, aim to obtain the preferred sequence for monitoring the SPIO labled cells; Identify the SPIO labled cells dynamics in vivo when transplanted in contralateral hemisphere of TBI model. We hope to find a easy and feasible technique and methods for future clinical trials of cellular therapy in TBI.Methods1. Culture and identification of BMSCs:BMSCs were derived from bone marrow aspirates of healthy adult rats by adherent culture method. Cells were identified by light microscope and transmission electron microscope; And detected the cell surface antigens (CD34, CD44, CD45, CD105); Induced the capability of neural differentiation of BMSCs by DMSO+BHA, and detected the expression of neuronspecific enolase (NSE), Glial Fibrillary Acidic Portein (GFAP) and nestin by immunocytochemistry technique.2. The labeled cells' identification and activity analysis:Check the intracellular iron by Prussian blue staining and electron microscopy of different concentrations of SPIO (Resovist) labled BMSCs, and analysis the cell activity by MTT growth curves.3. SPIO-labeled BMSCs were grafted stereotactically in the brain and tracked by MR in vivo:The brain injury model were developed as described by Feeney et al, and all of the rats were randomly and equally divided into 4 groups.Group A: TBI rats with BMSCs labeled by SPIO; Group B:TBI rats with BMSCs unlabeled by SPIO; Group C: negative control with only medium; Group D(sham group):only cut of the epicranium of rats without injury or transplatation. MR imaging were obtained on a 3.0T MR scanner to demonstrate the location, distribution and migration of the labled BMSCs at 1 day,1,2,4 weeks after injection by measuring Turbo Spin Echo (T1,T2), T2*-weighted GRE and susceptibility weighted imaging(SWI) sequence. Neurologic examinations (balance beam test) were performed at some time-points, such as 1 day,1 week, 2 weeks, and 4 weeks after the onset of TBI until sacrifice, aim to evaluate the effect of grafted cells. Finally after histological examination verified labeled cells in the brain's survival and migration.Results1. P3 generation of rat BMSCs demonstrated uniform distribution of the spindle or fiber-like cells; And showed the morphology of two different cell types under transmission electron microscope; Immunocytochemical staining results of BMSCs' CD molecules exhibited positive expression of CD44 and CD105 and negative expression of CD34 and CD45; The BMSCs were culture in nerve-induced liquid for 6 hours, the cells showed neuron-like cell under inverted phase contrast microscope, and express NSE, GAFP and Nestin when given immunocytochemical staining.2. The BMSCs were labled by the Resovist,, scattered brown granules can be seen in the cytoplasm of the cells under inverted phase contrast microscope; Positive express on Prussian Blue staining; many vesicles of iron particles can be seen in the cytoplasm under transmission electron microscope. The MTT test show that 14μg～112μg Fe/ml has no damage on cell activity.3. Neurological function score showed that A group vs. C, B group vs. C group had statistically significant between each other 1 day,1 week,2 weeks and 4 weeks after transplantation,but A group vs. B had no statistically significant between each other at any time-point. The injected site of the experimental animals MR results showed low signal intensity on T1WI, T2WI, T2*WI and SWI sequences, the signal loss significantly on T2*WI and SWI, and the SWI is the most sensitive sequence. The SPIO labled cells can be shown more clearly to cross from the contralateral hemisphere via a transcallosal path toward the lesion site by SWI at 2w and 4w time-point, and the tissue Prussian blue staining also confirmed the labled BMSCs migrated to the opposite side via a transcallosal path.Conclusions1. Resovist can access the cytoplasm of BMSCs efficiently without the help of transfection media,and it has no significant impact on cell growth and activity in the range of 14μg/ml～112μg/ml iron concentration.2. SPIO labled cells have been shown to cross from the contralateral hemisphere via a transcallosal path toward the lesion site by MRI, especially the susceptibility-weighted imaging sequence, besides, the motor function of the models were improved.